Wolbachia infection status and molecular diversity in the species of tribe Tagiadini Mabille, 1878 (Lepidoptera: Hesperiidae) collected in China

Abstract Wolbachia, one of the most ubiquitous heritable symbionts in lepidopteran insects, can cause mitochondrial introgression in related host species. We recently found mito‐nuclear discordance in the Lepidopteran tribe Tagiadini Mabille 1878 from which Wolbachia has not been reported. In this study, we found that 13 of the 46 species of Tagiadini species tested were positive for Wolbachia. Overall, 14% (15/110) of Tagiadini specimens were infected with Wolbachia and nine new STs were found from 15 isolates. A co‐phylogenetic comparison, divergence time estimation and Wolbachia recombination analysis revealed that mito‐nuclear discordance in Tagiadini species is not mediated by Wolbachia, but Wolbachia acquisition in Tagiadini appears to have occurred mainly through horizontal transmission rather than codivergence.

to better understand the ecology, evolution and establishment of Wolbachia symbiosis (Twort et al., 2022).
The Tagiadini Mabille 1878 tribe (c.160 species) of Hesperiidae is one of the most species-rich tribe within the subfamily Pyrginae (c.650 species) (Evans, 1949;Ma, 2019).Our previous study (Shen et al., 2022) described the phylogenetic relationship of the tribe in China based on concatenated mitochondrial and nuclear genes and also noted a mito-nuclear discordance in the molecular phylogeny of the Tagiadini (Figure 1).The discordance may be caused by species misidentification, incomplete lineage sorting (ILS), pseudogenes, introgressive hybridization and/or Wolbachia infection (Funk & Omland, 2003;Jiang et al., 2018;Toews & Brelsford, 2012).We excluded some of these such as species misidentification and pseudogenes in previous study (Shen et al., 2022).So far, there have been no systematic studies on Tagiadini members harbouring Wolbachia (Shen et al., 2022).
In the present study, we characterized the Wolbachia in this butterfly tribe by MLST genotyping, and compared a co-phylogenetic relationship and the divergence time between Wolbachia and their Tagiadini hosts.Furthermore, we analysed the Wolbachia recombination and transmission patterns across Tagiadini species and revealed the potential effects of Wolbachia on variation and evolution of mtDNA.While Wolbachia has been investigated in many lepidopteran insects with a particular focus on infection status, sequence information, recombination or transmission patterns (Andersen et al., 2019;Arif et al., 2021;Bereczki et al., 2015;Dincă et al., 2019;Gaunet et al., 2019;Gislaine et al., 2018;Hinojosa et al., 2019;Jiang et al., 2014Jiang et al., , 2016Jiang et al., , 2018;;Silva-Brandão et al., 2021;Śliwińska et al., 2019;Yudina et al., 2016;Zhang et al., 2021), there are few studies that focus on the inter-specific level across a large area.This

| Samples collection, DNA extraction and Wolbachia MLST typing
We collected a total of 110 Tagiadini specimens representing 18 genera and 46 species from 38 local regions in China across the last 15 years (Figure 2 and Table S1).All specimens were caught in the field and preserved following dehydration in small envelopes.When the samples were sent to our laboratory, the abdomens of the butterflies were cut and preserved in 100% ethanol, which was subsequently held at −20°C until DNA extraction for molecular analysis.The preliminary species-level identification was based on traditional morphological characteristics and molecular markers (Shen et al., 2022).The DNA was extracted from the entire adult butterfly abdomens using a QIAamp DNA Mini kit (Qiagen, Hilden, Germany) following the manufacturer's instructions.

| Co-phylogenetic analysis
For the molecular phylogenetic constructions of Tagiadini species (the concatenated mitochondrial and nuclear genes), we obtained the mitochondrial genes COI and COII, as well as three variable domains of nuclear DNA (D3 region of 28S rDNA, V4 and V7 regions of 18S rDNA) from GenBank (Table S1).The GTR + G model was selected as the best-fit substitution model with PartitionFinder v2.1.1 (Lanfear et al., 2012) using the Bayesian Information Criterion (BIC).A maximum likelihood (ML) tree was constructed with the concatenated data using IQtree 1.4.2(Nguyen et al., 2015).To assess nodal support, we performed 1000 ultrafast bootstrap replicates with UFBoot and an SH-aLRT test with 1000 replicates (Hoang et al., 2018).The MLST database was searched for homologous sequences of newly obtained MLST and wsp genes.The Wolbachia MLST sequences were aligned with outgroups retrieved from the MLST database (Table S1) using Bioedit v. 7.0 (Hall, 1999).The NJ tree of Wolbachia MLST was constructed by Mega 7 (Kumar et al., 2016).
The genetic distance matrices of Wolbachia and hosts were compared with IBD using the Mantel test (Bohonak, 2002) performed on a pairwise node distance matrix of Wolbachia genotypes and host Tagiadini species to test for an association between matrices (Hall, 1999).Another test of phylogenetic congruence between butterflies and endosymbiont partners was undertaken with the Procrustean Approach to Cophylogeny (PACo; Balbuena et al., 2013).The analysis was performed in R with 100,000 permutations using packages VEGAN v.2.4.6 (Oksanen et al., 2017) andAPE v.4.1 (Paradis et al., 2004).

| Divergence time estimation and recombination analysis
We established a comparison between the divergence times of Wolbachia and the age of Tagiadini species divergence according to a molecular dating analysis of Wolbachia supergroups A and B (Gerth & Bleidorn, 2016).The divergence times of Tagiadini species were inferred with the relaxed-clock molecular dating estimation by BEAST 1.5.2 (Excoffier et al., 2005).The analysis was performed using the HKY model of nucleotide substitution and assumed with the Yule speciation method.The best-fit model was selected with PartitionFinder v2.1.1 (Lanfear et al., 2012)  Jong, 2016).Chains were run for 50 million generations, with the first 20% discarded as burn-in.The results were summarized with TRACER 1.5 (Fu & Li, 1993).
Gene recombination can interfere with and mislead the phylogenetic relationships of species.We detected recombination events with the MLST and wsp genes, to clarify whether horizontal transmission had occurred among these Wolbachia genotypes.
In order to investigate recombination events among Wolbachia strains from Tagiadini species, each MLST gene and wsp gene were detected using RDP3 (Martin et al., 2010).Seven methods (RDP, GENECONV, BootScan, MaxChi, Chimaera, SiScan and 3Seq) in program RDP3 were chosen to identify the recombinant sequences and recombination breakpoints.The potential recombination events can be detected by any of the methods listed above.
As recommended for this procedure, the breakpoint positions and recombinant sequences inferred from every potential recombination event were manually checked and adjusted following the phylogenetic and recombination signal analysis features available in RDP3.To visualize potential recombination events, ML trees for each MLST gene and wsp were constructed with three outgroups retrieved from the MLST database (Table S1) using IQtree 1.4.2(Nguyen et al., 2015).They were checked for their supergroup clustering in ML trees.A potential recombination event can be found from inconsistencies between gene trees (Baldo, Bordenstein, et al., 2006).

| Infection rates and diversity of Wolbachia
Tagiadini samples were screened for Wolbachia through the amplification of the wsp locus.Fifteen individuals in 13 species were positive for Wolbachia and were shown to be polymorphic for the infection despite limited sampling.The infection status and geographical distribution of each individual are listed in Figure 2 and Table S1.We characterized Wolbachia genotypes by MLST typing.
The genotypes based on the MLST loci are denoted as wAda, wCho,wCva1,wCva2,wCva3,wDst,wDpt,wDte,wMtr,wPpi,wPbo and wSdo (Table 1).Seven genotypes belong to Wolbachia supergroup A and five genotypes belong to supergroup B. Three MLST genotypes (wDst, wMtr and wAda) were identical to those available in Wolbachia MLST databases (https:// pubml st.org/ wolba chia).wDst was identical to ST-41 belonging to Wolbachia supergroup B, wMtr was identical to ST-19 belonging to Wolbachia supergroup A and wAda was identical to ST-374 belonging to Wolbachia supergroup A. We found nine new STs from 15 isolates.ST-41 and ST-19 are the core genotypes of Wolbachia in lepidopteran hosts worldwide and detected in various Lepidopteran species (Ilinsky & Kosterin, 2017).

| Comparison of Wolbachia and Tagiadini phylogenies
Wolbachia genotypes in this study were species specific except for a host switching event and a case of a species harbouring different genotypes.The genotype wDst was shared by two host species (Darpa striata and Ctenoptilum multiguttatum) sympatric in Mengla, Yunnan Province, southwest China (Figure 2), while three Ctenoptilum vasava specimens harboured three genotypes respectively (wCva1, wCva2 and wCva3).Additionally, the Cophylogenetic relationships were not congruent between Wolbachia genotypes and corresponding Tagiadini hosts (Figure 3).These

F I G U R E 2
The distribution of Tagianidi specimens collected in China.The sizes of the circles are directly proportional to the number of individuals analysed.Three colour presents the different infection status (blue: infected with Wolbachia supergroup A; orange: infected with Wolbachia supergroup B; white: uninfected).The black dots refer to the location of collection sites and the letters are the abbreviation of place names.For full site names and other details, see Table S1.
suggested an important mode of transmission in Tagiadini Wolbachia was horizontal transmission.The Mantel test of genetic distances also provided no evidence for congruence between Tagiadini species and Wolbachia genotypes (r = .223,p = .624)in support of likely horizontal transmission.Similarly, PACo indicated no significant correlation between the phylogeny of Tagiadini and that of its endosymbiont (PACo m 2 = 0.062, p = .217).

| Divergence time estimation and recombination analysis
The relaxed clock molecular dating was implemented in BEAST and estimated with the divergence ages of the Tagiadini.The   & Bleidorn, 2016).We found the youngest divergence between species at 1.88 Mya (1.22-2.63,95% HPD) and the oldest gap between Odontoptilum angulata and the other species at 28.75 Mya (39.5-24.84,95% HPD) (Figure 4).
The recombination analysis found that the wsp sequence of the wDpt from Darpa pteria is the recombinant between Wolbachia wCva2 detected from Ctenoptilum vasava and wMtr from Mooreana trichoneura (Figure S1).The polymorphic sites of the Wolbachia wsp alleles have a mosaic pattern, but not randomly distributed.The recombination events were confirmed with seven RDP3 algorithms (Table S3).ML trees for MLST genes and wsp gene were reconstructed with three outgroups separately (Figure 5 and Table S1).
Five of the 12 Wolbachia genotypes (wCva1, wPpi, wCho, wSdo and wDte) had inconsistent supergroup allocation among the five MLST gene trees as the potential recombination events.

| DISCUSS ION
We present the detailed analyses of Wolbachia in Tagiadini butterflies from China by screening.Thirteen species (28%, 13/46) tested positive for Wolbachia (from both supergroups A and B) and all of which are reported for the first time.The species incidence of Wolbachia in this taxon is low when compared with an overall estimate of around 80% for Lepidoptera species (Ahmed, Araujo-Jnr, et al., 2015).Fourteen percent (15/110) of Tagiadini specimens were infected with Wolbachia.Larger sample sizes across the full geographical range of the Tagiadini tribe are needed to confirm these differences and more population samples are needed to establish the incidence of Wolbachia within infected species.
In the cophylogenetic analysis of Tagiadini based on mtDNA and corresponding nDNA (Figure S2), Wolbachia was not detected in some species with mito-nuclear discordance (e.g. six species of genus Capila), while infected species with mito-nuclear congruence were also detected (two species of genus Ctenoptilum, two species of genus Pintara, Mooreana trichoneura and Tapena thwaitesi).The remaining species infected with Wolbachia show mito-nuclear discordance with closely related uninfected species.It may further complicate the interpretation.In this study, we only examined and explained the infection by present.Thus, there is not necessarily a direct association between mito-nuclear discordance in Tagiadini species and the presence of Wolbachia.
The other bacterial symbionts such as Rickettsia, Cardinium or Spiroplasma were likely responsible for this (Zakharov, 2015).
Additionally, incomplete lineage sorting presumably can also lead to discordance between mtDNA and nDNA, but our restricted sample size and the few substitutions detected in the nuclear markers tested here make it difficult to use these data to reconstruct fine-scale phylogenies.
The recombination analysis showed that wsp gene in wDpt was an approximate intragenic recombination using RDP3 (Figure S1), suggesting a likely horizontal transmission occurred in the Tagiadini Wolbachia strains.The wsp gene shows a heterogeneous pattern of amino acid diversity characteristic marked by four distinct hypervariable regions (HVRs) interspaced by conserved strings of amino acids (CRs) (Baldo et al., 2005).Variants at each HVR have been frequently exchanged across bacterial strains, generating highly chimeric proteins (Baldo et al., 2005(Baldo et al., , 2010)).
Our results indicate that the phylogenetic relationships of Tagiadini hosts and corresponding Wolbachia genotypes were not congruent (Figure 3).Meanwhile, the correlation of genetic distances between Wolbachia genotypes and their Tagiadini hosts was not found by Mantel test and PACo, supporting the route of horizontal transmission of Tagiadini Wolbachia.The divergence age of Wolbachia was compared with that of their butterfly species (Figure 4).The divergence time between Wolbachia supergroups A and B was estimated over 200 Mya (Gerth & Bleidorn, 2016), while Tagiadini speciation events were dated between 1.88 and 28.75 Mya.This implies that the cocladogenesis is impossible between Tagiadini Wolbachia and their hosts because the Wolbachia Although there is a growing number of studies indicating or proving horizontal transmission of Wolbachia among coleopteran hosts, mostly via common host plants or the foraging substrate such as dung (Kajtoch, 2022), the transmission pathways in Lepidoptera are unclear but could occur through feeding on common plants (Li et al., 2017;Sintupachee et al., 2006), sucking from the same mud-pools (Duplouy et al., 2020) and exposure to ectoparasitic mites (Gehrer & Vorburger, 2012;Jaenike et al., 2007), or parasitoids (Ahmed, Li, et al., 2015;Vavre et al., 1999).Since many butterfly larvae feed on plant tissue, and adults obtain nectar from flowers or tree sap, the close association of butterflies with plants might lead to transmission being mediated through the host plant (Sintupachee et al., 2006).The mud pools could provide suitable short-term environments for Wolbachia to survive and transfer to a new host niche (Duplouy et al., 2020).However, this remains to be tested (Duplouy et al., 2020).Many hymenopteran parasitoids attack lepidopteran hosts and many of these are generalist parasitoids that could mediate horizontal transmission (Apiwathnasorn, 2012).

| CON CLUDING REMARK S
This study provides a conservative estimate of Wolbachia prevalence (14%) of the butterfly tribe Tagiadini with a species incidence of 28%, given the restricted nature of our sampling.

1
Cophylogenetic analysis of Tagiadini based on mtDNA (left) and corresponding nDNA (right).Connecting lines highlight the same species.Note the discordance in some parts of the phylogeny.| 3 of 11 WEI et al. has led us to complete a comprehensive survey of Wolbachia across the Tagiadini.
using the Bayesian Information Criterion (BIC).The age ranges provided by Chazot et al. (2019) were utilized to establish the calibration points for the divergence between Hesperiidae and Hedylidae (81-114 Mya), as well as the age ranges between Hesperiinae and Heteropterinae (35-55 Mya).Additionally, we applied a recently documented fossil hesperiid species, Pamphilites abdita Scudder, 1875 to constrain the minimum stem age of subfamily Hesperiinae to 25 Mya (de divergence time was compared between Wolbachia supergroups based on genomic data with data from Tagiadini in this study (Gerth F I G U R E 3 The tangled evolutionary relationships of Tagiadini when compared to their Wolbachia genotypes showing incongruence between host topology (left) and supergroup A and B Wolbachia topology (right).The orange solid lines point to a host switching event and the blue solid lines indicate a species harbouring different genotypes.The dotted lines connect the host species to the infected Wolbachia genotypes.Numbers beside nodes are IQTREE ultrafast bootstrap and SH-aLRT values (left) and bootstrap values (right).The Wolbachia genotypes of Supergroups A are in blue and those of Supergroups B are in orange.Scale bars indicate the mean number of substitutions per site.TA B L E 1 Wolbachia MLST and wsp profiles for Tagiadini butterflies collected from China.

F
I G U R E 4 (a) Estimated divergence times of Wolbachia supergroups A and B based on Gerth and Bleidorn (2016), and (b) Bayesian Inference (BI) tree of mtDNA datasets for Tagiadini species using the uncorrelated lognormal relaxed clock in BEAST v1.5.2.Posterior probabilities of nodes are shown to the right of the node branch when higher than .95.The violet bars (b) indicate 95% highest posterior density interval (HPD) of the node ages.Asterisks indicate the illustrated species.The adults are depicted in dorsal (left) and ventral (right) aspects.The red dots are the calibration points for age ranges.

F I G U R E 5
Maximum likelihood trees for each MLST gene and the wsp gene.Numbers beside nodes are IQTREE ultrafast bootstrap and SH-aLRT values.The Wolbachia strains of supergroup A are in blue and those of supergroup B are in orange.
genotypes have not been passed down from their common ancestor or transferred via hybridization events between the Tagiadini species.Two examples of likely horizontal transmission are provided by wDst and wCva3.wDst is shared by Tagiadini species including Darpa striata and Ctenoptilum multiguttatum.These two butterflies are sympatric in Mengla, reflecting an opportunity for horizontal transmission (Figure2).wCva3 is shared by Ctenoptilum vasava, Satarupa nymphalis and Tapena thwaitesi, all collected from Yunnan province.